Figure 1.

The images of fluorophores observed with a microscope are blurred by the wave-like
properties of light. (a) The image of a single fluorophore (red circle) has a width greater than approximately
250 nm when viewed with visible light, despite the fact that the fluorophore itself
is only a few nanometers in size. The image of such a point emitter is called the
point-spread function (PSF). The position of the fluorophore in this case can be determined
by measuring the center position of the image, which is equivalent to the PSF in this
case. (b) When multiple fluorophores are located in close proximity, their images overlap and
it becomes difficult to distinguish the individual fluorophores from one another.
It is the width of the PSF that limits the ability of the microscope to resolve closely
spaced fluorophores. The fluorophore positions cannot be determined accurately in
this case.